A. Specific Aims* Periodontal disease is a well-characterized chronic inflammatory bone destructive disease induced by bacterial infection with the Gram-negative pathogen Porphyromonas gingivalis. The cellular composition of inflammatory bone lesions in human periodontal disease consists of T and B cells, macrophages and dendritic cells (69,82,93). In other inflammatory bone diseases these cells have been reported to contribute to the acceleration of bone resorption by the production of proinflammatory cytokines including tumor necrosis factor (TNF)-[unreadable], interleukin (IL)-1[unreadable], IL-6, and receptor activator of nuclear factor [unreadable]B ligand (RANKL) (54,94,104). In addition, macrophages contribute to bone resorption through their differentiation to osteoclasts (1). Osteoclasts play an important role in bone resorption and in vitro studies have established that osteoclast differentiation occurs through RANKL and proinflammatory cytokine dependent mechanisms (8,61,71). However the ability of infectious agents to stimulate osteoclast differentiation is not well defined (56). P. gingivalis induces the production of an array of proinflammatory cytokines in various host cells in vitro (45,125). We recently reported that P. gingivalis infected macrophages induce osteoclastogenesis in vitro by a TNF-[unreadable] dependent / RANKL-independent mechanism which was dependent on the innate immune receptor Toll-like receptor, TLR2 (118). We also reported that TLR2 but not TLR4, was crucial for P. gingivalis induced oral inflammatory bone loss in a mouse model (45). Additional studies have established that B and T cells and proinflammatory cytokines contribute to oral inflammatory bone loss in response to P. gingivalis (15,41,45). Despite these observations, the role of TNF-[unreadable] and RANKL in P. gingivalis mediated inflammatory oral bone loss in vivo is not known. In this project we will test the hypothesis that specific TLR2 responsive immune cells produce proinflammatory cytokines in response to infection with P. gingivalis, which contribute to inflammatory oral bone loss in vivo. The following Aims are proposed: Aim 1. To define the role of TNF-[unreadable] and RANKL in P. gingivalis-induced oral inflammatory bone loss in a mouse model. A. We will examine P. gingivalis induced inflammatory bone loss in C57BL/6 and TNFR-/- mice. We will examine what immune cells constitute the inflammatory lesion induced following P. gingivalis infection and the innate immune signaling receptors and cytokines (TNF-[unreadable] and RANKL) expressed by these cells. B. We will examine the contribution of RANKL to P. gingivalis induced oral bone loss using OPG in P. gingivalis infected C57BL/6 and TNFR-/- mice. Aim 2. To define which TLR2 responding immune cells contribute to P. gingivalis-induced oral inflammatory bone loss in a mouse model. We will define which immune cells contribute to the TLR2 signaling response induced following P. gingivalis challenge by performing adoptive transfer studies with macrophages in C57BL/6 and TLR2-/- mice. We propose that an interplay between host specific immune cells and their responses to P. gingivalis plays a critical role in chronic inflammatory bone loss induced by P. gingivalis. Our studies will define the role of TLR2 responsive immune cells in inflammatory bone loss in vivo following stimulation with P. gingivalis. Enhanced understanding of the roles of specific immune cells and pathways that participate in proinflammatory cytokine expression and osteoclastogenesis in response to bacterial infection will provide a promising avenue for novel therapies for chronic inflammatory bone disorders. *References numbers are found in the "Literature Cited" section of the original grant application.